Broadly neutralizing anfibodies (BNAb) to HIV-1 primarily target the conserved membrane proximal ectodomain region (MPER) of the viral gpl60 envelope protein. We have studied the HxB2 MPER segment in lipid environments by a combinafion of nuclear magnefic resonance (NMR), electron paramagnetic resonance (EPR) and surface plasmon resonance (SPR) methodologies. Structural analyses reveal a tilted N-terminal a-helix (aa 664-672) connected via a short hinge (673-674) to a fiat Cterminal helical segment (675-683), collecfively forming a metastable L-shaped structure immersed in the membrane. The 4E10 BNAb extracts buried W672 and F673 following inifial encounter with the surface embedded MPER while the 2F5 BNAb lifts up residues N-terminal to the hinge region, exposing L669 and W670. BNAb CDRH3 interactions with lipid appear critical for neutralizing activity of both BNAbs. These data have implicafions for vaccine design and suggest how BNAbs can perturb tryptophan residueassociated viral fusion involving the MPER. Here we shall examine how other BNAbs or newly created MPER-binding anfibodies induce conformafional change around W672 and F673 or elsewhere using several disfinct MPER segment sequences. We shall determine whether such structural changes upon anfibody binding are linked to viral neutralization. Moreover, specificity and diversity of antibodies arising during natural HIV-1 infecfion vs. elicited upon vaccinafion will be compared. Sensitive EPR residue depth and inter-residue distance measurements will allow for relatively rapid screening of detectable changes in MPER conformafion. Once identified by EPR, interacfion will be followed by detailed NMR analysis. How lipid constituents of the virosome, including cholesterol, affect the membrane-embedded structure of the MPER, or its ability to undergo conformafional changes upon anfibody binding, will be assessed. In addifion, lipid-enveloped nanoparticles as carriers of natively configured MPER segments, including those with a bioresorbable poly (lactide-co-glycolide)(PLGA) core harboring "universal" class II MHC binding epitopes for maximal sfimulafion of follicular CD4+ T cell help, will be tested in murine intradermal immunizafion studies aimed at eliciting BNAbs. These nanoparticles, created in Project 2, of differing size and surface characteristics, will be further armed with targeting and immune activating adducts to opfimize the magnitude of the specific immune response using modulators of hypoxia-adenosinergic inhibition defined in Project in Project 4. ELISA, BlAcore and antibody neutralizafion assays will assess the breadth of neutralizing responses.